内皮素及其受体拮抗剂对新生大鼠心室肌细胞起搏电流及其基因表达的影响

Effects of endothelin and endothelin receptor antagonist on funny current and its gene expression in neonatal rat ventricular myocytes

目的:运用膜片钳全细胞技术和实时定量聚合酶链式反应(PCR),研究内皮素-1(ET-1)及其受体拮抗剂对新生大鼠心室肌起搏电流(If)及其基因表达的影响。方法:分离1-3d新生大鼠的心室肌细胞;测定超极化激活的环核苷酸门控通道(HCN)亚型1、HCN2、HCN3和HCN4的表达情况;记录If并研究其特性。其次分别用不同浓度(0、1、10、100nmol/L)ET-1刺激细胞3h,用100nmol/LET-1刺激细胞不同时间(0、0.5、1、3、6h),用100nmol/LET-1加1000nmol/LETA拮抗剂(BE-18257B)或ETB拮抗剂(IRL-1038)刺激心肌细胞3h,观察ET-1对If和HCN的影响。结果:HCN1、HCN2、HCN3和HCN4在总HCN的表达中所占比例分别为(0.23±0.01)%、(83.58±0.04)%、(0.79±0.01)%、(15.44±0.01)%。全细胞膜片钳记录到超极化激活、4mmol/LCsCl可阻断If;10、100nmol/LET-1刺激3h后,HCN2分别增加0.1、2倍,HCN4增加0.1、0.5倍;相同浓度ET-1刺激不同时间后,HCN2增加0.3、1、5、5.1倍,HCN4增加0.1、0.6、2、2.1倍,而ET-1对HCN1和HCN3无显著影响;加BE-18257B后HCN2和HCN4表达明显降低,If减小;而IRL-1038对HCN表达和If无显著影响。结论:(1)新生大鼠心室肌HCN的表达以HCN2和HCN4为主,并有超极化激活的If;(2)ET-1使其HCN2和HCN4表达增加,If增大,其作用主要通过ETA受体实现。

AIM： To analyze the effect of endothelin - 1 （ET - 1 ） and ET - 1 receptor antagonist on funny current （I1） and its gene （hyperpolarization- activated cation channel, HCN） expression in neonatal rat ventricular myocytes. METHODS ： Fresh ventricular myocytes were isolated from 1 - 3 d rats. The expression of If gene was measured by real - time quantitative polymerase chain reaction （ real - time PCR）. If was recorded and studied through whole - cell patch clamp. The cardiomyocytes were stimulated by ET - 1 （0, 1, 10, 100 nmol/L） for 3 hours, or ET - 1 （100 nmol/L） for 0, 0. 5, 1,3, 6 hours or ET - 1 plus ETA - or ETB - receptor antagonist （ BE - 18257B or IRL - 1038）, respectively. RESULTS： HCN1, HCN2, HCN3, HCN4 represented （0. 23 ± 0. 01 ） %, （ 83.58 ±0. 04） %, （0. 79 ± 0. 01） %, （ 15.44 ±0. 01 ） % of total HCN mRNA, respectively. If was recorded. When cells were stimulated by ET - 1 （ 10, 100 nmol/L）, HCN2 was significantly increased by0. 1,2 times and HCN4 was increased by0. 1,0. 5 times. When cells were stimulated for 0. 5, 1,3 and 6 hours, HCN2 was significantly increased by 0. 3, 1,5, 5.1 times and HCN4 was increased by 0. 1,0. 6, 2, 2. 1 times. The treatment of the combination of ET - 1 plus BE - 18257B significantly decreased HCN2 and HCN4 level. However, HCN1 and HCN3 had no statistically significant change. I1 would be increased by ET - 1 and this effect was reverted by BE - 18257B. IRL - 1038 had no effects on I1 and HCN. CONCLUSION： （ 1 ） HCN2 and HCN4 represent a large amount of total HCN mRNA in neonatal rat ventricular myocytes. （2） I1 and the expression of HCN2 and HCN4 are increased by ET - 1, this effect is reverted by BE - 18257B.